|
|
|
|
|
|
|
||
Articles |
Chain Plays an Essential Role in Regulating Lymphoid Homeostasis
Division of Hematologic Products, Food and Drug Administration, Bethesda, Maryland 20892
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResults and DiscussionReferences |
|---|
chain (c), and it was previously shown that c-deficient mice exhibit severely compromised development and responsiveness to IL-2, IL-4, and IL-7. Nevertheless, these mice exhibit an age-dependent accumulation of splenic CD4+ T cells, the majority of which have a phenotype typical of memory/activated cells. When c-deficient mice were mated to DO11.10 T cell receptor (TCR) transgenic mice, only the T cells bearing endogenous TCRs had this phenotype, suggesting that its acquisition was TCR dependent. Not only do the CD4+ T cells from c-deficient mice exhibit an activated phenotype and greatly enhanced incorporation of bromodeoxyuridine but, consistent with the lack of c-dependent survival signals, they also exhibit an augmented rate of apoptosis. However, because the CD4+ T cells accumulate, it is clear that the rate of proliferation exceeds the rate of cell death. Thus, surprisingly, although c-independent signals are sufficient to mediate expansion of CD4+ T cells in these mice, c-dependent signals are required to regulate the fate of activated CD4+ T cells, underscoring the importance of c-dependent signals in controlling lymphoid homeostasis.
The common cytokine receptor
Flow Cytometric Analysis.
Splenic CD4+ T Cell Purification.
Cell Survival Assay.
Annexin V Staining.
TUNEL Method.
Intracellular Staining of Bcl-2.
In Vivo Bromodeoxyuridine (BrdU) Uptake and BrdU Staining.
Apparent Rates of Replication and Survival. chain (c)1 is the genetic defect in X-linked severe combined immunodeficiency (XSCID) (1) and is a shared component of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15 (2–5). In humans with XSCID, the lack of functional c results in profoundly diminished T cell development and an absence of natural killer (NK) cells. In contrast, B cell numbers are normal, although the B cells are nonfunctional with concomitant hypogammaglobulinemia. Mice in which the c gene has been targeted by homologous recombination exhibit a related, but somewhat different phenotype (6–8). Like humans with XSCID, they have hypoplastic thymuses and lack NK cells. Unlike humans with XSCID, conventional B cells are greatly diminished in the bone marrow and the spleens of both young and adult c-deficient mice, likely reflecting the key role of IL-7 as a pre-B cell growth factor in mice, but not in humans (5). In addition, these mice lacked visible gut-associated lymphoid tissue and peripheral lymph nodes were essentially absent. Although splenic T cells are diminished in number at 3 wk of age, CD4+ T cells, but not CD8+ T cells, dramatically accumulate in the spleen in an age-dependent manner. Moreover, the finding of CD4+ T cell infiltrates, particularly in the gut in association with colitis, suggested that the CD4+ T cells might be activated and involved in mediating the pathological changes found in these mice. Given that the mice are compromised in their ability to respond to c-dependent cytokines including IL-2, IL-4, and IL-7 (6–8), this accumulation of CD4+ T cells was unexpected. We now have performed studies to characterize further these CD4+ T cells to understand better the role of c in development of the immune response in vivo.
Materials and Methods
Top
Abstract
Materials and Methods
Results and Discussion
References
Mice and Genetic Analysis.
The c-deficient mice (7) used in these studies were back-crossed to either C57BL/6 (B6) (H-2b/b) or BALB/c mice (H-2d/d) (Jackson Laboratory, Bar Harbor, ME) for three/four generations. Since the c gene is localized on X chromosome, DO10 TCR transgenic male mice (H-2d/d), specific for ovalbumin (9), were mated with BALB/c c+/– heterozygous female mice. These matings yielded four genotypes of male mice (DO10–c+/Y, DO10+c+/Y, DO10–c–/Y, and DO10+c–/Y). Similarly, AND TCR transgenic male mice (H-2b/b), specific for cytochrome c (10, 11), were mated with B6 c+/– female mice. Mice were housed in microisolator cages under pathogen-free conditions. The mice were genotyped by PCR of tail DNA using the following primer pairs: to detect the wild-type c gene, 5'-CTTTATTGATAACGATCTATCCCTCACCC-3' and 5'CTCCACTCTGCAGAGTCTATGGAATCC-3'; to detect the c knockout gene, 5'-GCTGACAGCCGGAACACGGCGG-3' and 5'-GTGCAATCCATCTTGTTCAATGGCCG-3'; to detect the DO10 transgene, 5'-CAGGAGGGATCCAGTGCCAGC-3' and 5'-TGGCTCTACAGTGAGTTTGGT-3'; to detect the AND transgene, 5'-GACTTGGAGATTGCCAACCCATATCTAAGT-3' and 5'-TGAGCCGAAGGTGTAGTCGGAGTTTGCATT-3'.
Cells from thymus and spleen were stained and analyzed on a FACSort® (Becton Dickinson, San Jose, CA) using Lysis II software (7). For direct staining, the following conjugated antibodies were purchased from PharMingen (San Diego, CA): anti-CD4 Cy-Chrome (H129.19), anti-CD8 PE (53-6.7), anti-CD25 PE (7D4), anti-CD44 Cy-Chrome (IM7), anti-CD45R (B220) PE (RA3-6B2), anti-CD49d FITC (R1-2), anti-CD62L PE (MEL-14), anti-CD69 PE (H1.2F3), anti-CTLA-4 PE (UC104F10-11), anti-erythroid cells PE (TER-119), anti-Fas biotin (Jo2), anti-I-Ab FITC (AF6-120.1), anti-I-Ad PE (AMS-32.1), anti-TCR V
11 FITC (RR8-1), and anti-Qa2a biotin (1-9-9) (12). KJ1-26 mAb, anti-idiotype for DO10 TCR (13) (provided by Dr. B.J. Fowlkes), was purified from supernatants of hybridoma cells using protein G columns (Pharmacia, Uppsala, Sweden) and conjugated to FITC.
Since erythroblastoid cells and macrophages were increased in the spleens of c-deficient mice as compared with wild type mice, single cell suspensions from spleen were treated with ACK lysing buffer to remove red cells, stained with PE-conjugated anti-CD8 mAb (53-6.7, PharMingen) and PE-conjugated anti-erythroid mAb (TER-119, PharMingen) and anti-CD16/CD32 mAb (FcRIII/II receptor, 2.4G2, PharMingen), washed twice with 1 x PBS containing 1% BSA (PBS, 1% BSA), and applied to T cell enrichment columns (R&D Systems). The resulting cells from both wild-type and c-deficient mice were 88–95% pure CD4+ T cells (average 91.8%) as determined by flow cytometry.
Purified CD4+ T cells were cultured in RPMI-1640 media supplemented with 10% charcoal-treated fetal bovine serum (Cocalico Biological, Inc., Reamstown, PA), 2 mM glutamine, and antibiotics for 8 to 48 h. Cells were harvested and stained with 5 µg/ml of propidium iodide (PI) (Boehringer Mannheim) and analyzed by flow cytometry.
After cells were stained with anti-CD4 Cy-Chrome and washed twice with PBS, 1% BSA, cells were stained with annexin V FITC (R&D Systems) according to the instructions of the manufacturer. Cells were analyzed on a FACSort®.
The terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end–labeling (TUNEL) method was performed according to the instructions of the manufacturer (Boehringer Mannheim). Splenocytes were stained with anti-CD4 CyChrome and washed with PBS, 1% BSA. Cells were then fixed with 70% ethanol for 20 min at 4°C, treated with PBS containing 1% paraformaldehyde and 0.01% Tween-20 for 30 min at 25°C, washed once with PBS, and incubated with the fluoresceinlabeled TUNEL reaction mix for 60 min at 37°C. As a negative control, cells were incubated without TdT. Cells were then washed with PBS, 1% BSA and analyzed on a FACSort®.
Bcl-2 levels in CD4+ T cells were analyzed as previously described (14). In brief, splenocytes (1 x 106) were permeabilized in PBS, 1% BSA containing 0.04% saponin (saponin buffer) and incubated with anti-Bcl-2 mAb (clone 3F11, PharMingen, 2 µg/ml final concentration) or purified hamster IgG as a negative control (4°C for 30 min). Cells were washed twice with saponin buffer, incubated with anti-hamster IgG cocktail conjugated to FITC (clone G70-204, G94-56, G91140, PharMingen) (4°C for 30 min), washed once with saponin buffer and once with PBS, 1% BSA, then stained with antiCD62L PE/and anti-CD4 Cy-Chrome, and analyzed on a FACSort®.
8 to 12-wk-old c-deficient or littermate control mice were injected i.p. with 750 µg of BrdU (Sigma) in 250 µl of PBS at time 0 and again at 4 h. Control mice were injected with PBS. 16 h after the second injection, thymocytes and purified splenic CD4+ T cells were fixed with 70% ethanol for 30 min on ice, denatured with 2 N HCl containing 0.5% Triton X-100 for 30 min to produce single-stranded DNA, and neutralized with 0.1 M sodium tetraborate, pH 9.0 (Sigma). Cells were resuspended in PBS, 1% BSA containing 0.5% Tween-20, stained with anti-BrdU FITC (Becton Dickinson) for 30 min at 25°C, washed once with PBS, 1% BSA containing 0.5% Tween-20, resuspended in PBS, 1% BSA, and analyzed on a FACSort®.
Assuming that cells that have taken up BrdU at 20 h have divided once, the apparent replication rate/day, r, can be calculated from the equation: percent BrdU uptake/20 h x 24 h/d = 2r/(100 + r). Based on the data in Fig. 1 D, this yields values for r of 21.43%/d for c-deficient mice and 6.32%/d for wild-type mice. Assuming that no death occurs and that there is no new migration of cells from thymus to periphery (neither assumption is likely to be correct), we can estimate the number of cells that might exist after 63 d of growth (from 3–12 wk of age) as 1.3 x 106 x (1.2143)63 = 2.66 x 1011 CD4+ splenic T cells instead of 5.28 x 107 cells for c-deficient mice and 9.1 x 106 x (1.0632)63 = 4.31 x 108 cells instead of 1.67 x 107 for wild-type mice. Although not accurate, these calculations suggest that survival rates for the cells differ between the wild-type and c-deficient mice. Again, assuming no new migration from the thymus to periphery and a constant replication rate, the survival rate/day can be estimated as 
87% for c-deficient mice and 95% for wild-type mice, suggesting that cells from the c-deficient mice had an increased rate of cell death (see below).
|
|
Results and Discussion |
|---|
|
TopAbstractMaterials and MethodsResults and DiscussionReferences |
|---|
c-deficient mice have hypoplastic thymi and lack NK cells (5–8). However, peripheral T cells are generally not found in humans with XSCID (4), whereas peripheral CD4+ T cells are found in the cdeficient mice (5–8). Although these cells are diminished in numbers in c-deficient mice at 3 wk of age (6, 7), in most mice there is a dramatic age-dependent accumulation of these cells (7) (Fig. 1 A), even though the thymus remains small (7). Analysis using a series of different TCR Vβ-specific mAbs (Vβ2, Vβ3, Vβ4, Vβ5.1,5.2, Vβ6, Vβ8.1,8.2, Vβ10, and Vβ11) indicated that the CD4+ T cell accumulation was polyclonal (unpublished data). Interestingly, the splenic CD4+ T cells in c-deficient mice were increased in size (Fig. 1 B). The large size of the cells, coupled with the finding of CD4+ T cell infiltrates, particularly in the gut in association with colitis (5, 7), suggested that the CD4+ T cells might be activated and involved in mediating the pathological changes found in these mice. Therefore, we investigated the basis for the CD4+ T cell expansion.
A striking difference between splenic CD4+ T cells from c-deficient and wild type mice was that the cells from the c-deficient mice exhibited a CD62LlowCD69high phenotype typical of memory/activated cells, whereas those from wild-type mice exhibited a CD62LhighCD69low phenotype typical of naive cells (Fig. 1 C) (15). Interestingly, CD4+ T cells in mice deficient in IL-2 (16, 17), IL-2R (18), and IL-2Rβ (19) also exhibited activated phenotypes. Expression of CD49d (VLA-4 chain) and CD44 was slightly increased in c-deficient CD4+ T cells (data not shown), whereas no significant difference was seen in expression of IL-2R (CD25) (Fig. 1 C), or in CD40L, CD28, CTLA-4, or in Fas (data not shown). The lack of increase in these activation markers suggested that these cells were partially rather than fully activated and that c-dependent signals are required for completing T cell activation. In contrast with the different phenotypes in splenic T cells, CD4+CD8– thymocytes from c-deficient mice and wild type mice expressed similar levels of CD62L, CD69, and Qa-2a (data not shown).
The accumulation and activation of the CD4+ T cells appeared to result from a defect in T cells, because the defect was corrected in mice in which c expression was selectively reconstituted in T cells (transgenic mice in which c is under control of the CD3
enhancer and promoter) but not in mice in which c expression was selectively reconstituted in B cells (transgenic mice in which c is under control of the mb-1 promoter and Eµ heavy chain enhancer) (data not shown). A similar activated phenotype in Jak3-deficient mice was corrected when these mice were reconstituted with Jak3 under control of the Lck promoter (19a), a finding consistent with the similar phenotypes in mice and humans deficient in either c or Jak3 and with the conclusion that Jak3 is the major mediator of c-dependent signals (4, 5).
Next, we investigated whether the CD62LlowCD69high phenotype of the c-deficient splenic CD4+ T cells reflected active proliferation and found that bromodeoxyuridine (BrdU) uptake in these cells was greater than in wild-type mice (Fig. 1 D, left). In contrast, c-deficient thymocytes exhibited lower BrdU uptake than wild-type thymocytes (Fig. 1 D, right). Thus, there was more active cell division in the spleens than in the thymi of adult c-deficient mice, correlating with the activated phenotype of the c-deficient splenocytes. It is important to recognize that because these mice were not subjected to thymectomy, some splenic BrdU uptake could have occurred in thymocytes before CD4+ T cell migration to the periphery; however, because the thymi in c-deficient mice are very small (0.5–5% of littermate controls) and the BrdU uptake in thymocytes in these mice was lower than in wild-type mice, it is likely that the majority of BrdU uptake occurred in the periphery.
Based on the BrdU uptake studies, the c-deficient mice had an apparent replication rate much greater than wildtype mice (21.43%/d for c-deficient mice and 6.32%/d for wild-type mice; see Materials and Methods). Such an increased rate for the c-deficient mice should have yielded a much greater accumulation of cells than actually seen (Fig. 1 A), suggesting that CD4+ T cells from the c-deficient mice had an increased rate of cell death (see Materials and Methods). Indeed, when cultured without stimulation, purified splenic CD4+ T cells from c-deficient mice exhibited a higher level of death than cells from wild-type mice, with the greatest difference occurring in the first 8 h (Fig. 2 A). Increased apoptosis was confirmed both by TUNEL analysis (data not shown) and by staining with annexin V (Fig. 2 B), which allows the detection of apoptotic cells at an early stage because of its selective affinity for phospholipids, especially phosphatidylserine (PS), which is exposed on the cell surface in apoptotic cells, while normally PS is restricted to the inner cell membrane (20–22). The rapid increase in death seen at 8 h in c-deficient mice (Fig. 2 A) presumably is accounted for by the large number of cells that stained with annexin V at time 0 (Fig. 2 B), suggesting that splenic CD4+ T cells of c-deficient mice may be primed to die by apoptosis in vivo. Bcl-2 levels correlate with the survival of lymphoid cells (23, 24) and are induced by c-dependent cytokines that protect against apoptosis (25–28). Therefore, it was interesting that c-deficient splenic CD4+ T cells expressed very low levels of Bcl-2 (Fig. 2 C) (including both CD62Lhigh and CD62Llow subpopulations; data not shown), suggesting that c is required for maintaining the normal levels of Bcl-2 expression in both activated and naive peripheral CD4+ T cells.
|
c-deficient mice were already activated without exogenous stimulation. In the first model, the activated phenotype does not require TCR stimulation (TCR-independent activation model); instead, c-dependent signals are required for keeping mature CD4+ T cells in a naive stage and, that without such signals, these cells are activated nonspecifically. In the second model, peripheral CD4+ T cells in c-deficient mice respond to self- or environmental antigens (TCR-dependent activation model). We investigated these possibilities using mice expressing MHC class II–restricted transgenic TCRs specific for ovalbumin (DO10 mice) or cytochrome c (AND mice). In these experiments, the mice were not exposed to the specific antigens. In DO10 male mice deficient in c (DO10+c–/Y mice), far fewer splenocytes were found than in DO10–c–/Y mice (6.4 x 106 versus 79 x 106 cells/spleen) (Fig. 3 A). Analogous differences were also seen between AND+c–/Y and AND–c–/Y mice (2.7 x 106 versus 50 x 106 cells/ spleen). These data suggested that endogenous TCR expression might be important in regulating CD4+ T cell accumulation. Using the DO10+c–/Y mice, we compared the relative frequency of memory (CD62Llow) and naive (CD62Lhigh) phenotypes in splenic CD4+ T cells expressing transgenic (KJ1-26+) versus endogenous (KJ1-26–) TCRs. Approximately 70% of CD4+ T cells from DO10+c–/Y mice express the transgenic TCR (Fig. 3 B, fourth histogram), analogous to DO10+c+/Y mice (second histogram). Surprisingly, although relatively high numbers of KJ126+CD4+ T cells in DO10+c–/Y mice were CD62Lhigh (naive phenotype), similar to the finding for DO10+c+/Y mice (84.9% versus 84.6%) (Fig. 3 C, bottom), relatively few of the endogenous KJ1-26– CD4+ T cells in DO10+c–/Y mice were CD62Lhigh (10%), similar to the finding for DO10–c–/Y mice (8.3%) (Fig. 3 C, top). Thus, the CD62Llow phenotype correlated with the presence of endogenous TCRs. Analogous results were found in AND TCR transgenic c-deficient (AND+c–/Y) mice. In AND+c–/Y mice, 93% of transgenic TCR+ (V11+) CD4+ T cells were CD62Lhigh, similar to AND+c+/Y mice (94%). These results support the TCR-dependent activation model and suggested that TCR(s) expressed on expanding peripheral CD4+ T cells in c-deficient mice are specific for self-antigen or environmental antigens. The finding that over 80% of adult c-deficient mice (C57BL/6 background) develop an inflammatory bowel disease (data not shown) is consistent with the possibility of self-reactive CD4+ T cells in these mice.
|
c-deficient mice, it was therefore surprising that although splenic T cells exhibit diminished in vitro responsiveness to mitogens (5), there was TCRdependent activation and a marked expansion of CD4+ T cells in vivo. In addition to this in vivo proliferation, we also detected an augmented rate of apoptosis in c-deficient CD4+ T cells, a finding consistent with the known action of c-dependent cytokines as survival factors (25–28); nevertheless, it was evident that the overall rate of death was inadequate for the degree of proliferation. Although the CD4+ T cells were activated, they expressed only low levels of CD25, CTLA-4, and Fas. Therefore, it is conceivable that c-dependent signals are required to achieve induction of pathways that are required to negatively regulate immune responses. Thus, c-dependent signals are essential components of a carefully controlled homeostatic mechanism that regulates the fate of activated cells.
|
Acknowledgments |
|---|
Submitted: 6 August 1996
Revised: 22 October 1996
1Abbreviations used in this paper:
c, common cytokine receptor chain; PI, propidium iodide; TUNEL, terminal deoxynucleotide transferase (TdT)- dUTP nick end–labeling; XSCID, X-linked severe combined immunodeficiency.
|
References |
|---|
|
TopAbstractMaterials and MethodsResults and DiscussionReferences |
|---|
1 Noguchi M, Yi H, Rosenblatt HM, Filipovich AH, Adelstein S, Modi WS, McBride OW & Leonard WJ. Interleukin-2 receptor chain mutation results in X-linked severe combined immunodeficiency in human, Cell, 1993, 73, 147–157.[Medline]
2 Leonard WJ, Noguchi M, Russell RM & McBride OW. The molecular basis of X-linked severe combined immunodeficiency: the role of the interleukin-2 receptor chain as a common chain, c, Immunol Rev, 1994, 138, 61–86.[Medline]
3 Sugamura K, Asao H, Kondo M, Tanaka N, Ishii N, Nakamura M & Takesha T. The common -chain for multiple cytokine receptors, Adv Immunol, 1995, 59, 225–277.[Medline]
4 Leonard WJ. The molecular basis of X-linked severe combined immunodeficiency: defective cytokine receptor signaling, Annu Rev Med, 1996, 47, 229–239.[Medline]
5 Leonard WJ, Shores EW & Love PE. Role of the common cytokine receptor chain in cytokine signaling and lymphoid development, Immunol Rev, 1995, 148, 97–114.[Medline]
6 DiSanto JP, Müller W, Guy-Grand D, Fischer A & Rajewsky K. Lymphoid development in mice with a targeted deletion of the interleukin-2 receptor chain, Proc Natl Acad Sci USA, 1995, 92, 377–381.
7 Cao X, Shores EW, Hu-Li J, Anver MR, Kelsall BL, Russell SM, Drago J, Noguchi M, Grinberg A, Bloom ET et al.. Defective lymphoid development in mice lacking expression of the common cytokine receptor chain, Immunity, 1995, 2, 223–238.[Medline]
8 Ohno K, Suda T, Hashiyama M, Mantani A, Ikebe M, Miyakawa K, Moriyama M, Nakamura M, Katsuki M, Takahashi K et al.. Modulation of hematopoiesis in mice with a truncated mutant of the interleukin-2 receptor chain, Blood, 1996, 87, 956–969.
9 Murphy KM, Heimberger AB & Loh DY. Induction by antigen of intrathymic apoptosis of CD4+ CD8+ TCRlothymocytes in vivo, Science (Wash DC), 1990, 250, 1720–1723.
10 Kaye J, Hsu M-L, Sauron M-E, Jameson SC, Gascoigne NRJ & Hedrik SM. Selective development of CD4+T cells in transgenic mice expressing a class II MHCrestricted antigen receptor, Nature (Lond), 1989, 341, 746–749.[Medline]
11 Vasquez NJ, Kaye J & Hedrick SM. In vivo and in vitro clonal deletion of double-positive thymocytes, J Exp Med, 1992, 175, 1307–1316.
12 Ramsdell F, Jenkins M, Dinh Q & Fowlkes BJ. The majority of CD4+CD8–thymocyte are functionally immature, J Immunol, 1991, 147, 1779–1785.[Abstract]
13 Marrack P, Shimonkevitz R, Hannum C, Haskins K & Kappler J. The major histocompatibility complex– restricted antigen receptor on T cells. IV. An antiidiotypic antibody predicts both antigen and I-specificity, J Exp Med, 1983, 158, 1635–1646.
14 Veis DJ, Sentman CL, Bach EA & Korsmeyer SJ. Expression of the Bcl-2 protein in murine and human thymocytes and in peripheral T lymphocytes, J Immunol, 1993, 151, 2546–2554.[Abstract]
15 Swain SL, Bradley LM, Croft M, Tonkonogy S, Atkins G, Weinberg AD, Duncan DD, Hedrick SM, Dutton RW & Huston G. Helper T-cell subsets: phenotype, function and the role of lymphokines in regulating their development, Immunol Rev, 1991, 123, 115–144.[Medline]
16 Sadlack B, Merz H, Schorle H, Schimpl A, Feller AG & Horak I. Ulcerative colitis-like disease in mice with a disrupted interleukin-2 gene, Cell, 1993, 75, 253–261.[Medline]
17 Sadlack B, Löhler J, Schorle H, Klebb G, Haber H, Sickel E, Noelle RJ & Horak I. Generalized autoimmune disease in interleukin-2-deficient mice is triggered by an uncontrolled activation and proliferation of CD4+T cells, Eur J Immunol, 1995, 25, 3053–3059.[Medline]
18 Willerford DM, Chen J, Ferry JA, Davidson L, Ma A & Alt FW. Interleukin-2 receptor a chain regulates the size and content of the peripheral lymphoid compartment, Immunity, 1995, 3, 521–530.[Medline]
19 Suzuki H, Kündig TM, Furlonger C, Wakeham A, Timms E, Matsuyama T, Schmits R, Simard JJL, Ohashi PS, Griesser H et al.. Deregulated T cell activation and autoimmunity in mice lacking interleukin-2 receptor β, Science (Wash DC), 1995, 268, 1472–1476.
19 Thomis DC & Berg LJ. Peripheral expression of Jak3 is required to maintain T lymphocyte function, J Exp Med, 1997, 185, 197–206.
20 Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL & Henson PM. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages, J Immunol, 1992, 148, 2207–2216.[Abstract]
21 Andree HAM, Reutelingsperger CPM, Hauptmann R, Hemker HC, Hermens WT & Willems GM. Binding of vascular anticoagulant (VAC) to planar phospholipid bilayers, J Biol Chem, 1990, 265, 4923–4928.
22 Vermes I, Haanen C, Steffens-Nakken H & Reutelingsperger C. A novel assay for apoptosis: flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labeled annexin V, J Immunol Method, 1995, 184, 39–51.[Medline]
23 Linette GP & Korsmeyer SJ. Differentiation and cell death: lessons from the immune system, Curr Opin Cell Biol, 1994, 6, 809–815.[Medline]
24 Yang E & Korsmeyer SJ. Molecular thanatopsis: a discourse on the Bcl2 family and cell death, Blood, 1996, 88, 386–401.
25 Otani H, Erdos M & Leonard WJ. Tyrosine kinase(s) regulate apoptosis and bcl-2 expression in a growth factor-dependent cell line, J Biol Chem, 1993, 268, 22733–22736.
26 Deng G & Podack ER. Suppression of apoptosis in a cytotoxic T-cell line by interleukin 2-mediated gene transcription and deregulated expression of the protooncogene bcl-2, Proc Natl Acad Sci USA, 1993, 90, 2189–2193.
27 Boise LH, Minn AJ, June CH, Lindsten T & Thompson CB. Growth factors can enhance lymphocyte survival without committing the cell to undergo cell division, Proc Natl Acad Sci USA, 1995, 92, 5491–5495.
28 Akbar AN, Borthwick NJ, Wickremasinghe RG, Panayiotidis P, Pilling D, Bofill M, Krajewski S, Reed JC & Salmon M. Interleukin-2 receptor common -chain signaling cytokines regulate activated T cell apoptosis in response to growth factor withdrawal: selective induction of anti-apoptotic (bcl-2, bcl-xL) but not pro-apoptotic (bax, bclxS) gene expression, Eur J Immunol, 1996, 26, 294–299.[Medline]
29 Green DR & Scott DW. Activation-induced apoptosis in lymphocytes, Curr Opin Immunol, 1994, 6, 476–487.[Medline]
30 Abbas AK. Die and let live: eliminating dangerous lymphocytes, Cell, 1996, 84, 655–657.[Medline]
31 Lenardo MJ. Fas and the art of lymphocyte maintenance, J Exp Med, 1996, 183, 721–724.
32 Kearney ER, Pape KA, Loh DY & Jenkins MK. Visualization of peptide-specific T cell immunity and peripheral tolerance induction in vivo, Immunity, 1994, 1, 327–339.[Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| TABLE OF CONTENTS |
|
